LOCUS       sequence-494557-        5399 bp    DNA     circular SYN 15-DEC-2025
DEFINITION  N-term GST tag E2 variant selected for LUBAC.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 5399)
  TITLE     E2 Variants for Probing E3 Ubiquitin Ligase Activities
  JOURNAL   Unpublished
REFERENCE   2  (bases 1 to 5399)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Dec 15, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"; journalName: 
            "Unpublished"
COMMENT     Sequence Label: GST-tag LUBAC binding E2 variant D3LU-2
FEATURES             Location/Qualifiers
     source          1..5399
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     promoter        join(1..6,7..22,23..29)
                     /label=tac promoter
                     /note="strong E. coli promoter; hybrid between the trp and 
                     lac UV5 promoters"
     protein_bind    37..53
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     CDS             76..729
                     /codon_start=1
                     /product="glutathione S-transferase from Schistosoma 
                     japonicum"
                     /label=GST
                     /translation="MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKK
                     FELGLEFPNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRY
                     GVSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYDALDVVL
                     YMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQATFGGGDHPPK"
     CDS             736..756
                     /codon_start=1
                     /product="tobacco etch virus (TEV) protease recognition and
                     cleavage site"
                     /label=TEV site
                     /translation="ENLYFQG"
     primer_bind     complement(1267..1289)
                     /label=pGEX 3'
                     /note="pGEX vectors, reverse primer"
     primer_bind     complement(1434..1452)
                     /label=pBRforEco
                     /note="pBR322 vectors, upsteam of EcoRI site, forward 
                     primer"
     promoter        1520..1624
                     /gene="bla"
                     /label=AmpR promoter
     CDS             join(1625..1693,1694..2485)
                     /codon_start=1
                     /gene="bla"
                     /product="beta-lactamase"
                     /label=AmpR
                     /note="confers resistance to ampicillin, carbenicillin, and
                     related antibiotics"
                     /translation="MSIQHFRVALIPFFAAFCLPVFAHPETLVKVKDAEDQLGARVGYI
                     ELDLNSGKILESFRPEERFPMMSTFKVLLCGAVLSRVDAGQEQLGRRIHYSQNDLVEYS
                     PVTEKHLTDGMTVRELCSAAITMSDNTAANLLLTTIGGPKELTAFLHNMGDHVTRLDRW
                     EPELNEAIPNDERDTTMPAAMATTLRKLLTGELLTLASRQQLIDWMEADKVAGPLLRSA
                     LPAGWFIADKSGAGERGSRGIIAALGPDGKPSRIVVIYTTGSQATMDERNRQIAEIGAS
                     LIKHW"
     primer_bind     complement(1843..1862)
                     /label=Amp-R
                     /note="Ampicillin resistance gene, reverse primer"
     rep_origin      2656..3244
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3145..3164
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3398..3415
                     /label=L4440
                     /note="L4440 vector, forward primer"
     promoter        3488..3565
                     /gene="lacI"
                     /label=lacI promoter
     CDS             3566..4648
                     /codon_start=1
                     /gene="lacI"
                     /product="lac repressor"
                     /label=lacI
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
                     /translation="MKPVTLYDVAEYAGVSYQTVSRVVNQASHVSAKTREKVEAAMAEL
                     NYIPNRVAQQLAGKQSLLIGVATSSLALHAPSQIVAAIKSRADQLGASVVVSMVERSGV
                     EACKAAVHNLLAQRVSGLIINYPLDDQDAIAVEAACTNVPALFLDVSDQTPINSIIFSH
                     EDGTRLGVEHLVALGHQQIALLAGPLSSVSARLRLAGWHKYLTRNQIQPIAEREGDWSA
                     MSGFQQTMQMLNEGIVPTAMLVANDQMALGAMRAITESGLRVGADISVVGYDDTEDSSC
                     YIPPLTTIKQDFRLLGQTSVDRLLQLSQGQAVKGNQLLPVSLVKRKTTLAPNTQTASPR
                     ALADSLMQLARQVSRLESGQ"
     primer_bind     complement(3585..3604)
                     /label=LacI-R
                     /note="LacI, reverse primer"
     promoter        join(4697..4702,4703..4720,4721..4727)
                     /label=lac promoter
                     /note="promoter for the E. coli lac operon"
     protein_bind    4735..4751
                     /label=lac operator
                     /bound_moiety="lac repressor encoded by lacI"
                     /note="The lac repressor binds to the lac operator to 
                     inhibit transcription in E. coli. This inhibition can be 
                     relieved by adding lactose or 
                     isopropyl-beta-D-thiogalactopyranoside (IPTG)."
     primer_bind     4759..4775
                     /label=M13 rev
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     4759..4775
                     /label=M13 Reverse
                     /note="In lacZ gene. Also called M13-rev"
     CDS             4771..5298
                     /codon_start=1
                     /gene="lacZ fragment"
                     /product="LacZ-alpha fragment of beta-galactosidase"
                     /label=lacZ-alpha
                     /translation="MTMITDSLAVVLQRRDWENPGVTQLNRLAAHPPFASWRNSEEART
                     DRPSQQLRSLNGEWRFAWFPAPEAVPESWLECDLPEADTVVVPSNWQMHGYDAPIYTNV
                     TYPITVNPPFVPTENPTGCYSLTFNVDESWLQEGQTRIIFDGVGISLSTARCTNASGVR
                     QPSEAVVWLCRS"
     primer_bind     complement(4791..4808)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
     primer_bind     complement(4791..4807)
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     primer_bind     complement(4800..4822)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
ORIGIN
        1 ttgacaatta atcatcggct cgtataatgt gtggaattgt gagcggataa caatttcaca
       61 caggaaacag tattcatgtc ccctatacta ggttattgga aaattaaggg ccttgtgcaa
      121 cccactcgac ttcttttgga atatcttgaa gaaaaatatg aagagcattt gtatgagcgc
      181 gatgaaggtg ataaatggcg aaacaaaaag tttgaattgg gtttggagtt tcccaatctt
      241 ccttattata ttgatggtga tgttaaatta acacagtcta tggccatcat acgttatata
      301 gctgacaagc acaacatgtt gggtggttgt ccaaaagagc gtgcagagat ttcaatgctt
      361 gaaggagcgg ttttggatat tagatacggt gtttcgagaa ttgcatatag taaagacttt
      421 gaaactctca aagttgattt tcttagcaag ctacctgaaa tgctgaaaat gttcgaagat
      481 cgtttatgtc ataaaacata tttaaatggt gatcatgtaa cccatcctga cttcatgttg
      541 tatgacgctc ttgatgttgt tttatacatg gacccaatgt gcctggatgc gttcccaaaa
      601 ttagtttgtt ttaaaaaacg tattgaagct atcccacaaa ttgataagta cttgaaatcc
      661 agcaagtata tagcatggcc tttgcagggt tggcaagcca cgtttggtgg tggcgaccat
      721 cctccaaaat cggatgaaaa cctgtatttt cagggatcca tggcactgaa acgcattaac
      781 aaagaactga gcgatctggc acgtgatccg cctgcacaga ttagcgcagg tccggttggt
      841 gatgatatgt ttcattggca ggcaaccatt atgggtccga atgatagccc gtatcaaggt
      901 ggtgtttttt ttctgaccat tcactttccg accgactatc cgtttaaacc gcctaaagtt
      961 gcatttacca cgcgtattta tcatccgaac attaatagca atggcagcat ttgtctggat
     1021 attctgcgta gccagtggat ggcggcactg accattagca aagttctgct gagcattgca
     1081 agcctgctgg atgatccgaa tccggatgat cctctggttc cggaaattgc acgtatctat
     1141 aaaaccgatc gcgataaata caatcgcatt agccgtgaat ggacccagaa atatgcaatg
     1201 taatgagcgg ccgcatcgtg actgactgac gatctgcctc gcgcgtttcg gtgatgacgg
     1261 tgaaaacctc tgacacatgc agctcccgga gacggtcaca gcttgtctgt aagcggatgc
     1321 cgggagcaga caagcccgtc agggcgcgtc agcgggtgtt ggcgggtgtc ggggcgcagc
     1381 catgacccag tcacgtagcg atagcggagt gtataattct tgaagacgaa agggcctcgt
     1441 gatacgccta tttttatagg ttaatgtcat gataataatg gtttcttaga cgtcaggtgg
     1501 cacttttcgg ggaaatgtgc gcggaacccc tatttgttta tttttctaaa tacattcaaa
     1561 tatgtatccg ctcatgagac aataaccctg ataaatgctt caataatatt gaaaaaggaa
     1621 gagtatgagt attcaacatt tccgtgtcgc ccttattccc ttttttgcgg cattttgcct
     1681 tcctgttttt gctcacccag aaacgctggt gaaagtaaaa gatgctgaag atcagttggg
     1741 tgcacgagtg ggttacatcg aactggatct caacagcggt aagatccttg agagttttcg
     1801 ccccgaagaa cgttttccaa tgatgagcac ttttaaagtt ctgctatgtg gcgcggtatt
     1861 atcccgtgtt gacgccgggc aagagcaact cggtcgccgc atacactatt ctcagaatga
     1921 cttggttgag tactcaccag tcacagaaaa gcatcttacg gatggcatga cagtaagaga
     1981 attatgcagt gctgccataa ccatgagtga taacactgcg gccaacttac ttctgacaac
     2041 gatcggagga ccgaaggagc taaccgcttt tttgcacaac atgggggatc atgtaactcg
     2101 ccttgatcgt tgggaaccgg agctgaatga agccatacca aacgacgagc gtgacaccac
     2161 gatgcctgca gcaatggcaa caacgttgcg caaactatta actggcgaac tacttactct
     2221 agcttcccgg caacaattaa tagactggat ggaggcggat aaagttgcag gaccacttct
     2281 gcgctcggcc cttccggctg gctggtttat tgctgataaa tctggagccg gtgagcgtgg
     2341 gtctcgcggt atcattgcag cactggggcc agatggtaag ccctcccgta tcgtagttat
     2401 ctacacgacg gggagtcagg caactatgga tgaacgaaat agacagatcg ctgagatagg
     2461 tgcctcactg attaagcatt ggtaactgtc agaccaagtt tactcatata tactttagat
     2521 tgatttaaaa cttcattttt aatttaaaag gatctaggtg aagatccttt ttgataatct
     2581 catgaccaaa atcccttaac gtgagttttc gttccactga gcgtcagacc ccgtagaaaa
     2641 gatcaaagga tcttcttgag atcctttttt tctgcgcgta atctgctgct tgcaaacaaa
     2701 aaaaccaccg ctaccagcgg tggtttgttt gccggatcaa gagctaccaa ctctttttcc
     2761 gaaggtaact ggcttcagca gagcgcagat accaaatact gtccttctag tgtagccgta
     2821 gttaggccac cacttcaaga actctgtagc accgcctaca tacctcgctc tgctaatcct
     2881 gttaccagtg gctgctgcca gtggcgataa gtcgtgtctt accgggttgg actcaagacg
     2941 atagttaccg gataaggcgc agcggtcggg ctgaacgggg ggttcgtgca cacagcccag
     3001 cttggagcga acgacctaca ccgaactgag atacctacag cgtgagctat gagaaagcgc
     3061 cacgcttccc gaagggagaa aggcggacag gtatccggta agcggcaggg tcggaacagg
     3121 agagcgcacg agggagcttc cagggggaaa cgcctggtat ctttatagtc ctgtcgggtt
     3181 tcgccacctc tgacttgagc gtcgattttt gtgatgctcg tcaggggggc ggagcctatg
     3241 gaaaaacgcc agcaacgcgg cctttttacg gttcctggcc ttttgctggc cttttgctca
     3301 catgttcttt cctgcgttat cccctgattc tgtggataac cgtattaccg cctttgagtg
     3361 agctgatacc gctcgccgca gccgaacgac cgagcgcagc gagtcagtga gcgaggaagc
     3421 ggaagagcgc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat
     3481 aaattccgac accatcgaat ggcgcaaaac ctttcgcggt atggcatgat agcgcccgga
     3541 agagagtcaa ttcagggtgg tgaatgtgaa accagtaacg ttatacgatg tcgcagagta
     3601 tgccggtgtc tcttatcaga ccgtttcccg cgtggtgaac caggccagcc acgtttctgc
     3661 gaaaacgcgg gaaaaagtgg aagcggcgat ggcggagctg aattacattc ccaaccgcgt
     3721 ggcacaacaa ctggcgggca aacagtcgtt gctgattggc gttgccacct ccagtctggc
     3781 cctgcacgcg ccgtcgcaaa ttgtcgcggc gattaaatct cgcgccgatc aactgggtgc
     3841 cagcgtggtg gtgtcgatgg tagaacgaag cggcgtcgaa gcctgtaaag cggcggtgca
     3901 caatcttctc gcgcaacgcg tcagtgggct gatcattaac tatccgctgg atgaccagga
     3961 tgccattgct gtggaagctg cctgcactaa tgttccggcg ttatttcttg atgtctctga
     4021 ccagacaccc atcaacagta ttattttctc ccatgaagac ggtacgcgac tgggcgtgga
     4081 gcatctggtc gcattgggtc accagcaaat cgcgctgtta gcgggcccat taagttctgt
     4141 ctcggcgcgt ctgcgtctgg ctggctggca taaatatctc actcgcaatc aaattcagcc
     4201 gatagcggaa cgggaaggcg actggagtgc catgtccggt tttcaacaaa ccatgcaaat
     4261 gctgaatgag ggcatcgttc ccactgcgat gctggttgcc aacgatcaga tggcgctggg
     4321 cgcaatgcgc gccattaccg agtccgggct gcgcgttggt gcggatatct cggtagtggg
     4381 atacgacgat accgaagaca gctcatgtta tatcccgccg ttaaccacca tcaaacagga
     4441 ttttcgcctg ctggggcaaa ccagcgtgga ccgcttgctg caactctctc agggccaggc
     4501 ggtgaagggc aatcagctgt tgcccgtctc actggtgaaa agaaaaacca ccctggcgcc
     4561 caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc tggcacgaca
     4621 ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatgtaagt tagctcactc
     4681 attaggcacc ccaggcttta cactttatgc ttccggctcg tatgttgtgt ggaattgtga
     4741 gcggataaca atttcacaca ggaaacagct atgaccatga ttacggattc actggccgtc
     4801 gttttacaac gtcgtgactg ggaaaaccct ggcgttaccc aacttaatcg ccttgcagca
     4861 catccccctt tcgccagctg gcgtaatagc gaagaggccc gcaccgatcg cccttcccaa
     4921 cagttgcgca gcctgaatgg cgaatggcgc tttgcctggt ttccggcacc agaagcggtg
     4981 ccggaaagct ggctggagtg cgatcttcct gaggccgata ctgtcgtcgt cccctcaaac
     5041 tggcagatgc acggttacga tgcgcccatc tacaccaacg tgacctatcc cattacggtc
     5101 aatccgccgt ttgttcccac ggagaatccg acgggttgtt actcgctcac atttaatgtt
     5161 gatgaaagct ggctacagga aggccagacg cgaattattt ttgatggcgt tggaattagc
     5221 ttatcgactg cacggtgcac caatgcttct ggcgtcaggc agccatcgga agctgtggta
     5281 tggctgtgca ggtcgtaaat cactgcataa ttcgtgtcgc tcaaggcgca ctcccgttct
     5341 ggataatgtt ttttgcgccg acatcataac ggttctggca aatattctga aatgagctg
//
